Conventional analytical measures to characterize cell growth and type suffer from substantial weaknesses. By itself, cell morphology is an unreliable means of cell identification. Morphological features can not be observed consistently, are inadequate for definitive identification, or require painstaking microscopic examination. Immunoassays and DNA probe assays can provide sensitive and specific characterizations of cells. However, these techniques have multiple drawbacks. They require the use of reagents which are complex and difficult to prepare. The assays themselves are complicated and time-intensive to perform. Often, the narrow specificity of an assay precludes development of a test that will respond to all strains of a single cell species.
This invention seeks to overcome these deficiencies by exploiting the metabolic functions of organisms and to offer a tool for characterization of organism growth and type with significant advantages over alternative analytical measures.
During the growth of microorganisms and cells, the composition of the supporting growth medium is altered as nutrients are converted by the organisms into metabolic end products. For example, complex molecules such as carbohydrates and lipids are converted by the cell's metabolic processes into smaller molecules such as lactic acid, succinic acid, acetic acid, or bicarbonate. Polypeptides and proteins are converted by way of amino acids to ammonia and bicarbonate. Thus, as growth occurs, the hydrogen ion content of the media changes as the starting nutrients are converted into metabolic products.
While these effects have been widely exploited by microbiologists for detection and identification of microorganisms using various manual and automated testing devices, piezoelectric oscillators have not been used previously in conjunction with metabolic products of cells to improve detection and analysis capabilities.
Previous efforts to apply piezoelectric oscillator techniques to biological activity require a time-consuming preparatory step in which one or more receptor materials, often antigens or antibodies, are immobilized on the surface of the piezoelectric oscillator. During operation of the piezoelectric oscillator, the receptor material binds with the target cell or analyte in a highly selective manner. The analyte drawn from solution may be complementary antibodies, antigens, DNA, or whole cells. Addition of the analyte changes the crystal's resonant frequency (.DELTA.f), the magnitude of which is proportional to the solution analyte concentration. This approach is described in UK Patent Application No. 86307115.5 by Seiko.
However, preparation of these receptor-modified piezoelectric devices is procedurally complex and often difficult. Even careful preparation does not guarantee consistent results. Receptor reagents are expensive, can inactivate during the immobilization process, and can separate from the surface of the crystal after immobilization. (G. G. Giulbault, J. H. Luong, and E. Pursak-Sochaczewski, Biotechnology, vol. 7, pp. 349-351, 1989.). Known techniques also require either washing or drying of the QCM (quartz crystal microbalance) or separation of "free" or "bound" reagents before QCM measurement. As a result, receptor-modified piezoelectric methods are not suited to continuous real-time measurement of metabolite production. Finally, the usefulness of receptor-modified piezoelectric methods is limited by the specificity of the reagents. It is extremely difficult to find a single reagent that responds broadly to many different cell strains or types, and thus repeated testing to assess the presence of different cell types has been necessary.
There currently exists a clear need for a piezoelectric method to detect, measure and analyze the growth of organisms in a way that overcomes the deficiencies of the existing technology. The ideal method should provide means of continuous detection, be simple to operate, economical, and quick, and should be useful for testing in a variety of different growth media compositions. The method should not require an immobilized receptor or complex washing and separation steps. The method should enable simultaneous tests using uncomplicated reagents. Unlike immunoassays or DNA probe assays, the method should be broadly applicable to all strains derived from a given species. The method also should be capable of detecting particular cells and microbes in the presence of mixed flora without prior separation into pure cultures. Thus, real time monitoring of the mixed culture would be available. Additionally, the method should be computer compatible so as to conveniently provide for statistical analysis of accumulated data. A device and a system to achieve these ends is also needed.